Tightly regulated Ca2+ homeostasis is essential for establishment of regular cardiac rhythm. Even a slight increase in cytosolic Ca2+ concentration can trigger fibrillation-like contraction and cardiac standstill in vivo and in vitro. Through zebrafish phenotype-based suppressor screening, we have recently identified a small molecule that restores fibrillation-like contraction of the heart. Biochemical analyses suggest that this compound binds to and potentiates a mitochondrial Ca2+ transporter, VDAC2. These data suggest a novel function of mitochondria as a fine-tuner of Ca2+ handling in the regulation of cardiac rhythmicity. Encouraged by the success of zebrafish screening, we propose to establish high- throughput screening system for forward chemogenetics approach to identify novel genetic pathways critical for the regulation of human cardiac Ca2+ homeostasis and rhythmicity using newly developed human embryonic stem cell- derived cardiomyocytes (hESC-CMs) differentiation protocol. Bioengineering approach, zebrafish in vivo heart and isolated adult mouse cardiomyocytes will be used as models to validate the hit compounds. We specifically propose to; 1. Assay development: We will optimize modalities to induce fibrillation, timing of the compound treatment, and readout for HTS. Efsevin will serve as a positive control. 2. Pilot screening: Pilot screening will be conducted with hESC-CM. 3. Hit validation: Once hits are identified, they will be tested on three model system. The long-term goal of this project is to establish a novel mechanism and treatment of atrial fibrillation, which affects 1% of total population older than 60 and 5% of 75 and older, and poses a high risk to stroke and other systemic emboli.